87 Determining the impact of CO₂ concentration and pH on mouse pre-implantation embryo development

Optimizing pH offers the potential to improve culture conditions and embryo development. It is established via equilibrium of hydrogen carbonate and CO₂ and each can impact cellular function. To investigate each variable’s individual impact on mouse embryo development, we explored the effect of incubator CO₂ concentration on embryo development. We hypothesised higher CO₂ levels could improve embryo development by meeting increased metabolic demands, as long as media pH stays within an acceptable range. Six replicates of in vivo matured oocytes were collected from CF-1 mice following equine chorionic gonadotrophin and human chorionic gonadotrophin stimulation and were fertilized in vitro. Fertilization was confirmed by the presence of two pronuclei (83.5 ± 8.1). One-cell embryos were randomly divided into three treatments (n = 144 each) and cultured in an Embryoscope™ for 115 h at 5% CO₂, 6% CO₂, or 7% CO₂ (sea-level values). The incubator settings were adjusted to account for the 2000 m elevation and set to 6.4%, 7.5%, and 8.9% CO₂. All treatments were cultured in an in-house single step culture medium containing 25 mM hydrogen carbonate supplemented with 10% recombinant human albumin (Recombumin^®) under 5% O₂. The pH of the culture media was measured at 7.52, 7.34, and 7.20, respectively. The cleavage, Day 4 blastocyst and Day 5 hatching blastocyst rates were recorded and analysed using chi-squared. The morphokinetics of time to cleavage, start of compaction, blastocoel formation, blastocyst stage (blastocoel >50% of embryo), expansion, and hatching were recorded. All hatching blastocysts were fixed and stained with SOX2 and CDX2 for cell counts. Data underwent normality testing using the Shapiro–Wilk test and were analysed using one-way ANOVA or Kruskal–Wallis test, P < 0.05. There were no significant differences in cleavage (100, 99 ± 0.01, and 100), Day 4 blastocyst (91 ± 0.1, 89 ± 0.1, and 94 ± 0.1) and Day 5 hatching blastocyst rates (87 ± 0.1, 88 ± 0.1, and 92 ± 0.1). However, the 5% CO₂ group took longer to reach the cleavage stage (18.1 ± 1.1, 18.0 ± 2.2, and 17.9 ± 1.2 h; P = 0.0238). No significant difference was found in time to compaction (65.0 ± 3.4, 65.0 ± 3.5, and 65.2 ± 4.4 h). The 7% CO₂ group reached the start of blastocoel formation (81.9 ± 6.9, 82.3 ± 7.8, and 80.1 ± 7.0 h; P = 0.0069), blastocyst stage (85.4 ± 8.3, 85.7 ± 8.5, and 82.9 ± 7.3 h; P = 0.0011), start of expansion (87.0 ± 7.5, 87.9 ± 9.2, and 84.9 ± 8.0 h; P = 0.0022) and hatching stage (89.8 ± 8.7, 90.6 ± 10.7, and 87.9 ± 9.7 h; P = 0.0205) significantly faster than the other groups. Moreover, hatching blastocysts from the 7% CO₂ group had a significantly higher total cell number (101.2 ± 27.6, 102.0 ± 28.7, and 110.3 ± 27.2; P = 0.0238), while inner cell mass/trophectoderm ratio (0.09 ± 0.04, 0.09 ± 0.05, and 0.09 ± 0.05) did not differ between groups. The increase in incubator CO₂ concentration significantly affected the development of mouse pre-implantation embryos, as indicated by changes in morphokinetics and total cell number. This suggests higher CO₂ levels may have a positive effect on pre-implantation embryo development. However, further experiments are necessary to determine the individual impacts of CO₂ concentration and pH, as these variables may be confounding each other and masking their true effects on embryo development.